The investigator will define the molecular interactions that drive RNA rearrangements during activation of the spliceosome for the catalysis of pre-mRNA splicing. The spliceosome is the macromolecular machine responsible for removing introns from pre-messenger RNAs, in preparation for their translation. It consists of five small nuclear RNAs, which are thought to catalyze the splicing reaction, and more than 60 proteins. Roughly speaking, the five RNAs can be thought of as having the following functions: one is an assembly factor (Ui), one negatively regulates catalytic activity (U4), one is a specificity factor (U5), and two constitute the active site (U2 and U6). This study will focus on the active site RNAs, particularly U6. Of the many splicing proteins, Prp8 is the largest (280 kDa), the most conserved (>60 percent identical between yeast and humans) and is most closely linked to the active site. They have discovered that Prp8 controls the unwinding of intermolecular helices between U4 and U6 RNAs, a necessary step in catalytic activation of the spliceosome. The molecular mechanism of this regulation will be studied in Aim 1. U6 RNA undergoes extensive changes in secondary structure during its incorporation into the spliceosome, and during the subsequent activation of the spliceosome for catalysis. The ability of an RNA to undergo such large changes in structure is unusual, and we believe it requires specialized RNA elements as well as assistance from proteins (i.e., RNA chaperones). Aim 2 will focus on the function of splicing factor Prp24, a prime candidate for a U6 RNA chaperone, in unwinding of the U4/U6 complex during spliceosome activation. After U4 RNA is displaced, U6 base-pairs with U2 RNA to form the catalytic core of the spliceosome. Little is known about how this secondary structure forms, or how it folds into a catalytically active tertiary structure. In Aim 3, they propose to identify new mutations in U6 and U2 RNAs that will enable genetic and biochemical analyses of this essential step in the splicing cycle.